Remedy for autoimmune diseases

ABSTRACT

Disclosed is use of nonsteroidal anti-estrogen compounds such as toremifene citrate as active ingredient for treating autoimmune diseases.

This application is a divisional of Ser. No. 07/192,990, filed, Nov. 16,1998, now U.S. Pat. No. 6,184,253 which is a divisal of Ser. No.08/844,846 filed Apr. 22, 1997, now U.S. Pat. No. 5,886,049, which is acontinuation of Ser. No. 08/448,348 filed Apr. 25, 1995, now aband whichis a 371 of PCT/JP93/01543, filed Oct. 26, 1993 which claims priorityfrom Japan Patent Appln 4-310,772 filed Oct. 27, 1992 which is adivisional of Ser. No. 09/516,435 Mar. 1, 2000.

TECHNICAL FIELD

The present invention relates to use of nonsteroidal anti-estrogencompounds (hereinafter referred to as nonsteroidal anti-estrogens) suchas toremifene, expected as a remedy for autoimmune diseases.

The autoimmune diseases include collagen diseases and the like. In lightof affected parts by the diseases, there are mentioned, for example,degenerative diseases of supporting tissues and connective tissues;autoimmune degenerative diseases of salivary glands, particularlySjögren's disease; autoimmune degenerative diseases of kidneys,particularly systemic lupus erythematodes and glomerulonephritis;autoimmune degenerative diseases of joints, particularly rheumatoidarthritis; and autoimmune degenerative diseases of blood vessels such asgeneralized necrotizing angitis and granulomatous angitis; and multiplesclerosis.

BACKGROUND ART

Immunosuppressants, nucleic acid antagonists, antimetabolites, etc., areused in the medicinal treatment of autoimmune diseases today.Anti-inflammatory agents, anticoagulants, etc., are also used in thesymptomatic therapies of the diseases. The effects of these agents are,however, not yet sufficient.

It is known that the immunosuppressants have side effects of provokingdiabetes, renal disorders, infectious diseases, etc. Also the use of thenucleic acid antagonist or antimetabolite is frequently accompanied byside effects such as hepatic disorders and medullary disorders. Thus themedicinal treatment of autoimmune diseases is so far very insufficient.

It has been demanded to develop a remedy for autoimmune diseases whichacts on the immune system and which has a function mechanism differentfrom that of conventional drugs for the diseases and less serious sideeffects.

DISCLOSURE OF INVENTION

After intensive investigations made for the purpose of finding theabove-described remedy, the present inventors have found thatnonsteroidal anti-estrogens have an excellent therapeutic effect on theautoimmune diseases and thus, based on this finding, completed thepresent invention.

The present invention relates to a remedy for autoimmune diseases whichcomprises as active ingredient a nonsteroidal anti-estrogen or apharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows survival times of animals (NZB×NZW F1 mice:B/W F1 mice)which accepted different doses of toremifene.

BEST MODE FOR CARRYING OUT THE INVENTION

The nonsteroidal anti-estrogen compounds usable in the present inventionare those having a triphenyl C₂-C₅ alkene or triphenyl C₂-C₅ alkaneskeleton. Preferably, they are C₂-C₅ alkenes or C₂-C₅ alkanes havingthree phenyl substituents at the 1-position and 2-position, wherein anyof the phenyl groups may have a substituent such as a mono- or di-loweralkyl (C₁-C₃) amino lower alkoxy (C₁-C₃) group, or a hydroxyl group, orthe alkyl group in the above alkenes or alkanes may have a substituentsuch as a halogen.

Examples of these compounds include toremifene (JP-B-4 19973), tamoxifen(JP-B-59 21861), 4-hydroxytamoxifen (JP-A-54 44644), 3-hydroxytamoxifen(JP-A-57 122049) and N-demethyltoremifene or 4-hydroxytoremifene (JP-A-3163015). Toremifene is particularly preferred. It is well-known thatthese compounds have an anti-neoplastic effect (see Cancer Chemotherapyand Pharmacology, 17, 109-113 (1986) and the above-mentioned patentpublications).

The pharmaceutically acceptable salts thereof include, for example,hydrochlorides, sulfates, citrates, tartrates and phosphates.

Drugs usable in combination with the nonsteroidal anti-estrogens in themedicinal treatment of autoimmune diseases include glucocorticoids (e.g.prednisolone, prednisone, cortisol). Prednisolone is preferred.

The glucocorticoids themselves have an effect of treating the autoimmunediseases. The nonsteroidal anti-estrogens or a pharmaceuticallyacceptable salt thereof according to the present invention concomitantwith the glucocorticoids synergistically improve the effect of treating.

The remedy of the present invention particularly exhibits an excellentremedial effect on systemic lupus erythematodes.

Therefore the present invention relates to the following:

(i) a remedy for autoimmune diseases which comprises as activeingredient a nonsteroidal anti-estrogen or a pharmaceutically acceptablesalt thereof;

(ii) a remedy recited in (i), wherein the nonsteroidal anti-estrogen isa compound having a triphenyl C₂-C₅ alkene or triphenyl C₂-C₅ alkaneskeleton;

(iii) a remedy recited in (i) or (ii), wherein the active ingredient istoremifene or a pharmaceutically acceptable salt thereof;

(iv) a remedy recited in (i) or (ii), wherein the autoimmune diseasesare collagen diseases, autoimmune degenerative diseases of kidneys suchas nephritis, particularly glomerulonephritis, and autoimmunedegenerative diseases of blood vessels, salivary glands and joints;

(v) a remedy recited in (i) or (ii), wherein the autoimmune diseases aresystemic lupus erythematodes; and

(vi) a remedy recited in (i) or (ii) for concomitant use with aglucocorticoid.

The pharmaceutical composition of the present invention is administeredorally, parenterally or intravenously.

Usually, a pharmaceutically effective amount of the active ingredient isused in combination with a suitable medicinal carrier or otherauxiliaries. The term “pharmaceutically effective amount” herein meansan amount capable of exhibiting the intended pharmacological activitywithout causing unfavorable side effects. The accurate amount varies ineach case depending on various factors such as administration methods,individual natures of the patients and situations in which the patientaccepts the remedy and, as a matter of course, structures of derivativesto be administered.

Dose of the active ingredient for adult is usually 10 to 1000 mg/day,preferably 20 to 500 mg/day, more preferably 30 to 300 mg/day.

In the case of the concomitant use, dose of the glucocorticoid for adultis 1 to 100 mg/day, preferably 2 to 60 mg, and that of the nonsteroidalanti-estrogen or the pharmaceutically acceptable salt thereof for adultis 10 to 700 mg/day, preferably 20 to 500 mg/day, more preferably 30 to300 mg/day.

The medicinal carrier or other auxiliaries generally usable incombination with the active ingredient according to the presentinvention may be any of solid and liquid ones and usually selected inconsideration of an administration route. Examples of the solid carrierinclude lactose, sucrose, gelatin and agar, and those of the liquidcarrier include water, syrup, peanut oil and olive oil. Other suitablecarriers and auxiliaries known by those skilled in the art are alsousable. The active ingredient according to the present invention can becombined with the carrier or other auxiliaries to form any of variousacceptable preparations such as tablets, capsules, suppositories,liquid, emulsion and powder.

In the preparations of the remedy of the present invention, the amountof the nonsteroidal anti-estrogen or the pharmaceutically acceptablesalt thereof can widely vary depending on the preparation, etc. Usually,the amount is 0.01˜100% by weight, preferably 0.1˜70% by weight, and thebalance contains the medicinal carrier or other auxiliaries.

MRL/Mp-lpr/lpr mice spontaneously develop a lethal glomerulonephritis,angitis, sialadenitis, polyarthritis, etc., concurrently with thedeposition of an immune complex with age. Therefore, they are widelyused as experimental models for human systemic lupus erythematodes,Sjögren's disease, rheumatoid arthritis and autoimmune angitis such asmultiple arteritis.

The present invention will be explained referring to examples onsuppression of lymphadenopathy glomerulonephritis, angitis, sialadenitisand arthritis of MRL/Mp-lpr/lpr mice with the nonsteroidal anti-estrogencompound according to the present invention.

The nonsteroidal anti-estrogen such as toremifene and thepharmaceutically acceptable salt thereof according to the presentinvention exhibit an excellent remedial effect on degenerative diseasessuch as autoimmune diseases, for example, systemic lupus erythematodes.

EXAMPLE 1

Treatment of Spontaneous Autoimmune Diseases of MRL/Mp-lpr/lpr Mice ByAdministration of2[4-(Z)-4-chloro-1,2-diphenyl-1-butenyl]phenoxy-N,N-dimethylethylamineCitrate (Toremifene Citrate)

Eight-week old female MRL/Mp-lpr/lpr mice (Clea Japan, Inc.) were usedin this examination. Toremifene citrate (JP-B-4 19973) was suspended incarboxymethylcellulose to prepare a 0.5% suspension. This compound (100mg/kg) was orally administered to each mouse once a day for 13 weeks.

(A) Inhibition of Swelling of Spleen and Lymph Node of MRL/Mp-lpr/lprMice With Toremifene Citrate

Repeated oral administration of 100 mg/kg of toremifene citrate once aday for 13 weeks inhibited the swelling of the spleen and lymph node ofeach mouse (see Table 1).

The spleen and lymph nodes of the MRL/Mp-lpr/lpr mice are seriouslyswollen with age due to the presence of the lymphoproliferation gene(lpr). The lpr codes for the Fas antigen in each mouse. However, in theMRL/Mp-lpr/lpr mice, an abnormality of the genes disturbs the expressionof the Fas antigen. As a result, autoreactive T-cells are not subjectedto negative selection through the Fas antigen in the thymus and appearin the peripheral tissues to cause the swelling of the lymphoid organsand autoimmune symptoms. The presence of the autoreactive T-cells wasconfirmed also in the autoimmune diseases of human beings, such asrheumatoid arthritis.

The results of this study indicated that the nonsteroidal anti-estrogencompounds such as toremifene citrate are capable of inhibiting theappearance of the autoreactive T-cells, thereby suppressing the swellingof spleen and lymph node to treat the autoimmune diseases.

TABLE 1 Effect of toremifene citrate¹⁾ on swelling of spleen and lymphnode MRL/Mp-lpr/lpr mice Group Number of animals$\frac{{Spleen}\quad {weight}^{4)}}{{Body}\quad {weight}}$

$\frac{{Lymph}\quad {node}\quad {weight}^{5)}}{{Body}\quad {weight}}$

Control²⁾ 11   2.34 ± 0.74³⁾ 6.77 ± 1.70 Toremifene 12 1.38 ± 1.06 3.11± 1.43 citrate treatment ¹⁾Toremifene citrate (100 mg/kg) was orallyadministered to 8-week old mice once a day for 13 weeks. ²⁾Only 0.5%carboxymethylcellulose was given to the mice of the control group.³⁾Standard deviation${\quad^{4)}{Spleen}\quad {node}\quad {{weight}/{body}}\quad {weight}} = {\frac{{Weight}{\quad \quad}{of}{\quad \quad}{spleen}}{{Body}{\quad \quad}{weight}{\quad \quad}{of}\quad {mouse}} \times 100}$

${\quad^{5)}{Lymph}\quad {node}\quad {{weight}/{body}}\quad {weight}} = {\frac{{Weight}{\quad \quad}{of}{\quad \quad}{lymph}\quad {node}}{{Body}{\quad \quad}{weight}{\quad \quad}{of}\quad {mouse}} \times 100}$

(B) Suppression of Renal Disorder of MRL/Mp-lpr/lpr Mouse WithToremifene Citrate

An autopsy was performed on the mice of the control group and thetoremifene citrate treated group after the completion of theadministration to examine their kidneys pathohistologically. The bloodurea nitrogen (BUN) of the serum in each group was examined to confirmchanges in the renal function. As shown in Table 2, toremifene citrateameliorated the glomerulonephritis and healed the renal function in theMRL/Mp-lpr/lpr mice.

The glomerulonephritis of the MRL/Mp-lpr/lpr mice is caused by thedeposition of immunocomplexes. Also in the case of the autoimmunediseases such as systemic lupus erythematodes (SLE) of human, thepatients suffer from glomerulonephritis concurrent with the depositionof the immunocomplex. The results indicated that the nonsteroidalanti-estrogen compounds such as toremifene citrate are effectiveremedies for the degenerative diseases of the kidney, such as the SLEwith renal syndrome and glomerulonephritis.

TABLE 2 Improvement of renal function and amelioration ofglomerulonephritis of MRL/Mp-lpr/lpr mice with toremifene citrate Numberof BUN Group animals Glomerulonephritis¹⁾ (mg/dl)²⁾ Control 11   2.4 ±0.7³⁾ 43.1 ± 23.9 Toremifene 12 1.2 ± 0.7 24.6 ± 4.9  citrate treatment

1) The kidney was fixed in 10% buffered formalin, and then paraffinsections thereof were prepared by an ordinary method to prepare HE andPAS stained specimens. The extent of the disorder of the renal glomeruliwas scored and classified into the following groups:

0 (no disorder),

1 (slight disorder),

2 (medium disorder), and

3 (heavy disorder).

Twenty-five renal glomeruli were observed for each mouse and the averagethereof was calculated.

2) The BUN was determined with a Fuji Dry Chem Analyzer.

3) Standard deviation.

(C) Inhibition By Toremifene Citrate of Sialadenitis, Angitis andArthritis of MRL/Mp-lpr/lpr Mice

The salivary gland, renal blood vessel and knee joint of each mouse inthe control group and the toremifene citrate treated group werehistopathologically examined.

As shown in Table 3, toremifene citrate prevented the mice from beingattacked by sialadenitis, angitis and arthritis.

These results indicated that the nonsteroidal anti-estrogen compoundssuch as toremifene citrate and tamoxifen citrate can be used as theremedy for autoimmune sialadenitis (Sjögren's disease), autoimmunearthritis (chronic articular rheumatism) and autoimmune angitis(necrotizing angitis and granulomatous angitis).

TABLE 3 Effect of toremifene citrate for preventing MRL/Mp-lpr/lpr micefrom being attacked by sialadenitis, angitis and arthritis Number ofGroup animals Sialadenitis¹⁾ Angitis¹⁾ Arthritis¹⁾ Control 11 2.2 ± 0.6²⁾ 2.1 ± 0.7 1.6 ± 0.9 Toremifene 12 0.9 ± 0.8 0.9 ± 0.8 0.4 ± 0.5citrate treatment

1) The salivary gland, kidney and knee joint were fixed in 10% bufferedformalin, and then paraffin sections thereof were prepared by anordinary method to prepare HE and PAS stained specimens. The extent ofthe disorder was scored and classified into the following groups:

0 (no disorder),

1 (slight disorder),

2 (medium disorder), and

3 (heavy disorder).

2) Standard deviation.

EXAMPLE 2

Effect of Concomitant Use of Toremifene Citrate With Glucocorticoid onMRL/Mp-lpr/lpr Mice

Twelve-week old female MRL/Mp-lpr/lpr mice were used in the examination.Thirty miligrams per kg or 15 mg/kg of toremifene citrate (TOR) wasorally administered to each mouse twice a day for 9 weeks from the 12thweek to the 21st week. A glucocorticoid (prednisolone), 8, 4 and 2mg/kg/day, were subcutaneously administered to mice once a day as apositive control drug. The concomitant use of tremifene with theglucocorticoid was also carried out according to the same regimen asabove. The kidney was taken out from each mouse the day after thecompletion of the whole administration period and fixed in a PLPfixative. Frozen sections were made from the fixed kidney and used foran immunostaining with an anti-Mac-2 monoclonal antibody (Hybritec Inc.,San Diego, USA). The number of Mac-2 positive cells (activatedmacrophages) invading each of 10 to 20 glomeruli of the kidney, which ishereinafter referred to as Mac 2 number, was counted under a microscopyto determine an average Mac 2 number per glomerulus. The degree ofsevereness of glomerulonephritis was estimated in terms of the averageMac 2 number (n=13 for each group). Table 4 shows the results.

TABLE 4 Suppression of glomerulonephritis of MRL/Mp- lpr/lpr mice byconcomitant use of toremifene citrate with glucocorticoid Group Mac 2number Control 7.5 ± 1.5 Toremifene citrate (TOR) 30 mg/kg 6.2 ± 1.0 15mg/kg 6.5 ± 1.2 Prednisolone (P) 8 mg/kg 5.8 ± 0.8 4 mg/kg 7.9 ± 0.7 2mg/kg 9.4 ± 1.0 Control 11.3 ± 1.2 Prednisolone (P) 4 mg/kg 9.1 ± 1.4 2mg/kg 7.7 ± 1.0 P 4 mg/kg & TOR 30 mg/kg (concomitant use) 4.1 ± 0.5* P4 mg/kg & TOR 15 mg/kg (concomitant use) 4.3 ± o.5* P 4 mg/kg & TOR 7.5mg/kg (concomitant use) 3.5 ± 0.5* P 2 mg/kg & TOR 30 mg/kg (concomitantuse) 3.6 ± 0.7* P 2 mg/kg & TOR 15 mg/kg (concomitant use) 2.8 ± 0.5* P2 mg/kg & TOR 7.5 mg/kg (concomitant use) 4.3 ± 0.6* *P < 0.01 (t-test)

All the groups treated by concomitant use of toremifene citrate (TOR)with prednisolone (P) exhibited significant decrease in Mac 2 number ascompared with the control and the prednisolone treated group. On theother hand, the prednisolone treated group and the toremifene citratetreated group did not exhibit any significant decrease in Mac 2 numberas compared with the control. The results of these tests indicates thatthe concomitant use of the both drugs synergistically suppresses theglomerulonephritis.

EXAMPLE 3

Comparison of Survival Time

NZB×NZW mice (B/W F1 mice) were used as a pathological model ofautoimmune diseases (systemic lupus erythematodes). Effect of toremifenecitrate on the survival time of the animals was investigated.

Experimental Animals

F1-hybrids of NZB (female) and NZW (male) mice (B/W F1 mice): Importedfrom Bomholtgaard, Denmark at the age of five weeks.

Test Groups and Doses

Control (male): administration polyethyleneglycol (peg) 3 times a weekper os

Control (female): administration peg 3 times a week per os

Toremifene citrate 30 mg/kg/day: administration 70 mg/kg in polyethyleneglycol solution 3 times a week per os to female NZB×NZW F1 mice

Toremifene citrate 3 mg/kg/day: administration 7 mg/kg in polyethyleneglycol solution 3 times a week per os to female NZB×NZW F1 mice

The survival time of the animals in different test groups is presentedin FIG. 1. All but two female control animals have died during thealmost two years' follow-up time. Fifty percents of the animals in thisgroup died before/at the age of 40 weeks, and 20% (4/20) were aliveafter one year.

In the male control group, five animals died during the first 24 weeks(not shown in FIG. 1) due to aggressive behaviour and thereby acquiredinfection. These five were excluded from the results. Forty-sevenpercents of the male control mice are still alive after almost twoyears' time.

In both toremifene treatment groups the life span of the animals haslengthened clearly when compared to the female control animals. In the 3mg/kg toremifene treatment group only one (1/20) animal had diedat/before the age of 40 weeks and three (3/20) animals in the 30 mg/kgtoremifene group.

After one year 80% and 85% of the animals were alive in the 3 mg/kg and30 mg/kg toremifene treated groups, respectively, which is nearer thepercentage of the male control animals (≈90%) than that of the femalecontrol group (20%).

Moreover, 25% (5/20) and 10% (2/20) of the animals are still alive afteralmost two years' time in the lower and higher toremifene dosage group,respectively.

The follow-up data of 60 female and 15 vale F1-hybrids of NZB×NZW F1mice (B/W F1 mice) show that toremifene treatment has clearly extendedthe life span of female mice.

EXAMPLE 4

Examples of preparations comprising the nonsteroidal anti-estrogen orthe pharmacologically acceptable salt thereof as active ingredient willbe given below, which by no means limit the preparations of the presentinvention.

Preparation Example 1

Formulation of prepared 200 mg tablet.

Toremifene citrate 20 mg Starch 85 mg Lactose 90 mg Magnesium stearate 5mg

Preparation Example 2

Formulation of prepared 200 mg tablet.

Tamoxifen citrate 20 mg Starch 85 mg Lactose 90 mg Magnesium stearate 5mg

What is claimed is:
 1. A method for treating autoimmune degenerativediseases of joints, comprising administering to a patient in needthereof a therapeutically effective amount of toremifene or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein said autoimmune degenerative disease of joints is rheumatoidarthritis.
 3. The method of claim 1, further comprising the concomitantadministration of glucocorticoid.
 4. The method of claim 2, furthercomprising the concomitant administration of glucocorticoid.